U.S. patent application number 11/324834 was filed with the patent office on 2006-07-06 for parallel preparation of high fidelity probes in an array format.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Robert G. Kuimelis, Glenn H. McGall.
Application Number | 20060147971 11/324834 |
Document ID | / |
Family ID | 36640935 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060147971 |
Kind Code |
A1 |
Kuimelis; Robert G. ; et
al. |
July 6, 2006 |
Parallel preparation of high fidelity probes in an array format
Abstract
The present invention provides massively parallel
oligonucleotide synthesis and purification for applications that
utilize large collections of defined high-fidelity oligonucleotides
(e.g., from about 10.sup.1 to about 10.sup.5 different sequences,
generally between 25-160 bases in length).
Inventors: |
Kuimelis; Robert G.; (Palo
Alto, CA) ; McGall; Glenn H.; (Palo Alto,
CA) |
Correspondence
Address: |
AFFYMETRIX, INC;ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3420 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC.
Santa Clara
CA
95051
|
Family ID: |
36640935 |
Appl. No.: |
11/324834 |
Filed: |
January 3, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60640317 |
Dec 31, 2004 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
427/2.11; 435/287.2; 435/6.1 |
Current CPC
Class: |
C40B 40/06 20130101;
B01J 2219/00596 20130101; B01J 2219/00427 20130101; B01J 19/0046
20130101; B01J 2219/00527 20130101; B82Y 30/00 20130101; B01J
2219/00608 20130101; C40B 80/00 20130101; B01J 2219/00675 20130101;
C40B 50/14 20130101; B01J 2219/00711 20130101; B01J 2219/00722
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 427/002.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Claims
1. A method of fabricating a plurality of oligonucleotides having
free 3'-hydroxyl groups from a high density oligonucleotide array,
said method comprising of a) providing a solid substrate comprising
a plurality of ribonucleotides attached thereto at a density, one
said ribonucleotide shown below ##STR15## wherein PG.sub.1 is
protecting group 1, PG.sub.2 is protecting group 2, B is naturally
or non-naturally occurring base, and said ribonucleotide is
attached to said substrate through the 5'-hydroxyl group; b)
selectively removing PG.sub.1 in pre-selected areas to provide a
plurality of free 3'-hydroxyl groups on said ribonucloetide; c)
reacting said free 3'-hydroxyl groups with a 2'-deoxyribonucleotide
having the structure ##STR16## wherein PG.sub.3 is protecting group
3 and RG is a reactive group to couple said 2'-deoxyribonucleotide
to said ribonucleotide to provide the structure ##STR17## d)
selectively removing PG.sub.3 from the 5'-hydroxyl of said
2'-deoxyribonucleotide in pre-selected areas to provide a plurality
of free 5'-hydroxyl groups; e) reacting said free 5'-hydroxyl
groups with additional 2'-deoxyribonucleotide having the structure
##STR18## to yield a product of the structure ##STR19## f)
repeating steps d and e one or more times to provide said
oligonucleotides attached to said solid substrate; g) removing
PG.sub.2 from one or more of said ribonucleotides to provide a free
2'-hydroxyl group on each of said one or more ribonucleotides; and
h) transesterifying each of said one or more ribonucleotides to
yield said solid substrate having a cyclic ester attached thereto
and free oligonucleotides, each oligonucleotide having a
3'-hydroxyl group and having the structure ##STR20##
2. A method according to claim 1 wherein PG.sub.2 is acetate.
3. A method according to claim 1 wherein PG.sub.3 or PG.sub.4 is a
photolabile protecting group.
4. A method according to claim 3 wherein said photolabile
protecting group is selected from the group consisting of NNPOC and
MBPMOC wherein said photolabile protecting group is attached to the
5'-hydroxyl group of said 2'-deoxyribonucleotide as depicted by
##STR21##
5. A method according to claim 1 wherein the 2'-deoxyribonucleotide
has a phosphoramidite reactive group as shown by ##STR22## wherein
R.sub.1 is selected from cyanoethyl, methyl, t-butyl,
trimethylsilyl or the like, and R.sub.2 and R.sub.3 are
independently selected from isopropyl, cyclohexyl or the like.
6. A method according to any of claims 1-5 wherein B of the
2'-deoxyribonucleotide is selected from the group consisting of G,
A, T, and C.
7. A method according to claim 1 wherein PG.sub.3 or PG.sub.4 is an
acid labile protecting group.
8. A method according to claim 7 wherein PG.sub.3 or PG.sub.4 is
dimethoxytrityl (DMT).
9. A method according to claim 8 wherein the DMT group is removed
in selected areas by exposure to acid generated by a photoacid
generator in the presence of electro magnetic radiation of an
appropriate wavelength in the presence of an acid scavenger.
10. A method according to claim 9 wherein said acid scavenger is
selected from the group consisting of organic bases and polymeric
bases.
11. A method according to claim 10 wherein said acid scavenger is a
polymeric base.
12. A method according to claim 1 wherein said plurality of
oligonucleotides comprises between 10.sup.1 to 10.sup.5 sets of
different sequences, each distinct set of sequences comprising a
set.
13. A method according to claim 1 wherein said oligonucleotides are
between about 80 to about 160 nucleotides.
14. A method according to claim 1 wherein said density is between
200-2000 pmol/cm.sup.2.
15. A method according to claim 1 wherein said transesterification
is accomplished by raising the pH of the solution to between 9 and
12.
16. A method according to claim 1 wherein said tranesterification
is initiated by the addition of metal ions.
17. A method according to claim 1 wherein PG.sub.2 is selected from
the group consisting of FPMP, CEE, TBDMS and TOM.
18. A method according to claim 1 wherein said oligonucleotides are
probes.
19. A method according to claim 1 wherein said oligonucleotides are
primers.
20. A method according to claim 1 wherein between steps d and e
unreacted 5'-hydroxyls are capped.
21. A method of purifying a set of oligonucleotides, comprising the
steps of p1 a) providing a solid substrate comprising a plurality
of ribonucleotides attached thereto at a density, one said
ribonucleotide shown below ##STR23## wherein PG.sub.1 is protecting
group 1, PG.sub.2 is an alkalai resistant protecting group, B is a
naturally or non-naturally occurring base, and said ribonucleotide
is attached to said substrate through the 5'-hydroxyl group; b)
selectively removing PG.sub.1 in pre-selected areas to provide a
plurality of free 3'-hydroxyl groups on said ribonucloetide; c)
reacting said free 3'-hydroxyl groups with a 2'-deoxyribonucleotide
having the structure ##STR24## wherein PG.sub.3 is DMT, B is a
naturally or non-naturally occurring base in which the exocyclic
amine groups are protected with alkalai labile protecting groups,
and RG is a reactive group to couple said 2'-deoxyribonucleotide to
said ribonucleotide to provide the structure ##STR25## d)
selectively removing PG.sub.3 from the 5'-hydroxyl of said
2'-deoxyribonucleotide in pre-selected areas to provide a plurality
of free 5'-hydroxyl groups; e) reacting said free 5'-hydroxyl
groups with an additional 2'-deoxyribonucleotide having the
structure ##STR26## wherein PG.sub.4 is DMT, to yield a product of
the structure ##STR27## f) repeating steps d and e one or more
times to provide said oligonucleotides attached to said solid
substrate; g) deprotecting said set of oligonucleotides while said
oligonucleotides are still attached to said substrate by subjecting
said oligonucleotides to alkaline conditions, wherein said alkaline
conditions remove said alkalai labile protecting groups acting to
protect said exocyclic amines and in addition cleave depurinated
DNA, leaving a 3'-end of the cleaved depurinated strand attached to
the substrate and releasing a truncated fragment; h) washing the
solid support to remove said released truncated fragments and
protecting groups, leaving full length oligonucleotides having a
DMT group on the 5'-hydroxyl group and truncated oligonucleotides
without the 5'-DMT group; i) removing PG.sub.2 from one or more of
said ribonucleotides to provide a free 2'-hydroxyl group on each of
said one or more ribonucleotides; j) transesterifying each of said
one or more ribonucleotides to yield said solid substrate having a
cyclic ester attached thereto and a mixture of full length
oligonucleotides having 5'-DMT groups and free 3'-hydroxyl groups
and having the structure ##STR28## and truncated fragments lacking
the DMT group; k) applying the mixture to hydrophobic
oligonucleotide purification resin to isolate only those
oligonucleotides having the 5'-DMT group to yield full length
oligonucleotides; and l) removing the 5'-DMT group to provide sets
of full length oligonucleotides having both 5'- and 3'-hydroxyl
groups.
22. A method according to claim 21 wherein said PG.sub.2 is
selected from the group consisting of FPMP, CEE, TBDMS, TOM and a
photo labile protective group.
23. A method according to claim 22 wherein said PG.sub.2 is
selected from the group consisting of FPMP and CEE.
24. A method according to claim 23 wherein PG.sub.2 is removed with
mild acid.
25. A method according to claim 22 wherein said PG.sub.2 is
selected from the group consisting of TBDMS and TOM.
26. A method according to claim 25 wherein PG.sub.2 is removed by
exposure to fluoride ions.
27. A method according to claim 22 wherein said PG.sub.2 is a photo
labile protecting group removed by exposure to electromagnetic
radiation of about 310 nm and up.
28. A method according to claim 27 wherein said photolabile
protecting group is selected from the group consisting of NNPOC and
MBPMOC.
29. A method for massively parallel oligonucleotide probe synthesis
and release of said oligonucleotides from an array of probes on a
solid substrate, said method comprising the steps of: providing a
solid substrate; attaching a plurality of linkers to the substrate,
each said linker comprising a cleavable moiety, wherein said
cleavable moiety is activatable only at a distinct set of
conditions and wherein activation of said cleavable moiety disrupts
the linker to allow release of the polymer, to provide a substrate
with a plurality of attached linkers; attaching a first monomer to
at least one of said plurality of attached linkers to provide an
attached first monomer; attaching a second monomer to a least one
of said attached first monomers or said plurality of attached
linkers to provide an attached second monomor; attaching a third
monomer to a least one of said attached first monomer, said second
monomer or said plurality of attached linkers to provide an
attached third monomer; repeating said steps of attaching monomers
until the desired array of polymers is complete; and subjecting the
array to the distinct set of conditions to release polymers from
said array.
30. A method according to claim 1 wherein said desired array has
between 10.sup.1 to 10.sup.5 oligonucleotides of different
sequences and between about 80-160 bases in length.
31. A method according to claim 1 wherein said release
oligonucleotides have authentic 3'-hydroxy termini upon exposure to
said distinct set of conditions or are further processed to have
authentic 3' hydroxyl termini.
32. A method according to claim 1, further comprising the use of
the photoprotective groups NPPOC or NNPOC to provide suittable
primer purity and quantity.
33. A method according to claim 1 wherein DMT-based photoresist
groups are used to provide fidelity primers.
34. A method according to claim 1 wherein ink-jet based in situ
oligonucleotide synthesis is used to provide the oligonucleotide
prove.
35. A method according to claim 1 wherein synthesis is initiated
with a reverse-orientation RNA monomer that contains an orthogonal
2'-OH protecting group.
36. A method according to claim 7 wherein following conventional
3'.fwdarw.5' probe synthesis, the 2'-OH protecting group is removed
to allow base-induced intramolecular transesterification.
38. A method according to claim 8 wherein the rate of
transesterification is enchanced by raising the pH of the aqueous
solution to pH 9-12 or by the addition of particular metal ions.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the fabrication
of oligonucleotides such as probes and primers using
oligonucleotide array technology. The present invention relates to
massively parallel oligonucleotide synthesis and purification for
applications that utilize large collections of defined
high-fidelity oligonucleotides.
BACKGROUND OF THE INVENTION
[0002] PCR techniques are well-established and widely used across
various segments of life-science research, diagnostics, etc. An
increasingly important trend in the application of PCR is the
ability to multiplex the reaction, which requires, in addition to
the usual thermal cycling equipment and enzyme, sets of carefully
designed oligonucleotide primers (or probes). Oligonucleotide
primers are traditionally prepared by the solid-supported
phosporanidite approach, either on controlled-pore glass, polymeric
support or membrane support.
[0003] Following oligonucleotide assembly, the support is typically
treated with a deprotection reagent to remove protecting groups and
to cleave the oligonucleotide from the support in a single step.
Due to the high stepwise efficiency of the solid-supported
phosphoramidite approach, it is often not necessary to rigorously
purify short oligonucleotides (25-40 mers) destined for use as PCR
primers. More often, simple ethanol precipitation or cartridge
separation is used to "desalt" the primer and remove small
molecular-weight components.
[0004] Although careful purification is atypical, some means of
identity and purity confirmation (i.e., QC) are normally required
and the collection of such data is considered good lab practice.
Primer confirmation can usually be accomplished by high-throughput
analytical techniques such as MALDI-TOF mass spectrometry andlor
capillary gel electrophoresis. Conventional small-scale
solid-supported oligonucleotide synthesis methods (flow-through
column, membrane, 96-well plate) produce enough primer for
thousands of PCR reactions.
SUMMARY OF THE INVENTION
[0005] Methods are provided for generating high numbers of
oligonucleotides as probes and primers using oligonucleotide array
technology to provide oligonucleotides, probes and primers.
[0006] In particular, methods are also provided for fabricating a
plurality of oligonucleotides having free 3'-hydroxyl groups from a
high density oligonucleotide array. In a preferred embodiment,
synthesis is initiated with a reverse-orientation RNA monomer that
contains an orthogonal 2'-OH protecting group. Following
conventional 3'.fwdarw.5' probe synthesis, the 2'-OH protecting
group is removed to allow base-induced intramolecular
transesterification. The transesterification reaction causes
release of the synthesized probe with an authentic 3'-hydroxy
functionality, while the 2',3'-cyclic phosphate remains attached to
the solid support.
[0007] Another disclosed method has the steps of providing a solid
substrate; attaching a plurality of linkers to the substrate, each
said linker having a cleavable moiety, wherein the cleavable moiety
is activatable at a distinct set of conditions and wherein
activation of the cleavable moiety disrupts the linker to allow
release of anything joined to the linker at the site of the
cleavable moiety, to provide a plurality of attached linkers;
attaching a first monomer to at least one of said plurality of
linkers to provide an attached first monomer; attaching a second
monomer to a least one of said attached first monomers or said
attached plurality of linkers to provide an attached second
monomer; attaching a third monomers to a least one of said attached
first monomer, second monomers or plurality of linkers to provide
an attached third monomer; repeating said step of attaching a
monomer until the desired array of polymers is complete and
subjecting the array to the distinct set of conditions to provide
release of polymers from the array.
DESCRIPTION OF THE DRAWINGS
[0008] "FIG. 1" depicts the transesterification reaction causing
release of the synthesized probe with an authentic 3'-hydroxy
functionality, while the 2',3'-cyclic phosphate remains attached to
the solid support.
[0009] "FIG. 2" shows two exemplary monomers used in the process
depicted in FIG. 1.
[0010] "FIG. 3" depicts a purification scheme to obtain full length
probes with authentic termi.
DETAILED DESCRIPTION OF THE INVENTION
[0011] A. General
[0012] The present invention has many preferred embodiments and
relies on many patents, applications and other references for
details known to those of the art. Therefore, when a patent,
application, or other reference is cited or repeated below, it
should be understood that it is incorporated by reference in its
entirety for all purposes as well as for the proposition that is
recited.
[0013] As used in this application, the singular form "a," "an,"
and "the" include plural references unless the context clearly
dictates otherwise. For example, the term "an agent" includes a
plurality of agents, including mixtures thereof.
[0014] An individual is not limited to a human being but may also
be other organisms including but not limited to mammals, plants,
bacteria, or cells derived from any of the above.
[0015] Throughout this disclosure, various aspects of this
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0016] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, biochemistry, and
immunology, which are within the skill of the art. Such
conventional techniques include polymer array synthesis,
hybridization, ligation, and detection of hybridization using a
label. Specific illustrations of suitable techniques can be had by
reference to the example herein below. However, other equivalent
conventional procedures can, of course, also be used. Such
conventional techniques and descriptions can be found in standard
laboratory manuals such as Genome Analysis: A Laboratory Manual
Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells:
A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular
Cloning: A Laboratory Manual (all from Cold Spring Harbor
Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.)
Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical
Approach" 1984, IRL Press, London, Nelson and Cox (2000),
Lehninger, Principles of Biochemistry 3.sup.rd Ed., W.H. Freeman
Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5.sup.th
Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein
incorporated in their entirety by reference for all purposes.
[0017] The present invention can employ solid substrates, including
arrays in some preferred embodiments. Methods and techniques
applicable to polymer (including protein) array synthesis have been
described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.
5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,
5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215,
5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734,
5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324,
5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860,
6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT
Applications Nos. PCT/US99/00730 (International Publication No. WO
99/36760) and PCT/US01/04285 (International Publication No. WO
01/58593), which are all incorporated herein by reference in their
entirety for all purposes.
[0018] Patents that describe synthesis techniques in specific
embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216,
6,310,189, 5,889,165, and 5,959,098. Nucleic acid arrays are
described in many of the above patents, but the same techniques are
applied to polypeptide arrays.
[0019] Nucleic acid arrays that are useful in the present invention
include those that are commercially available from Affymetrix
(Santa Clara, Calif.) under the brand name GeneChip.RTM.. Example
arrays are shown on the website at affymetrix.com.
[0020] The present invention also contemplates many uses for
polymers attached to solid substrates. These uses include gene
expression monitoring, profiling, library screening, genotyping and
diagnostics. Gene expression monitoring and profiling methods can
be shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135,
6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses
therefore are shown in U.S. Ser. Nos. 10/442,021, 10/013,598 (U.S.
Patent Application Publication 20030036069), and U.S. Pat. Nos.
5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799
and 6,333,179. Other uses are embodied in U.S. Pat. Nos. 5,871,928,
5,902,723, 6,045,996, 5,541,061, and 6,197,506.
[0021] The present invention also contemplates sample preparation
methods in certain preferred embodiments. Prior to or concurrent
with genotyping, the genomic sample may be amplified by a variety
of mechanisms, some of which may employ PCR. See, for example, PCR
Technology: Principles and Applications for DNA Amplification (Ed.
H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A
Guide to Methods and Applications (Eds. Innis, et al., Academic
Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res.
19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17
(1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S.
Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675,
and each of which is incorporated herein by reference in their
entireties for all purposes. The sample may be amplified on the
array. See, for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No.
09/513,300, which are incorporated herein by reference.
[0022] Other suitable amplification methods include the ligase
chain reaction (LCR) (for example, Wu and Wallace, Genomics 4, 560
(1989), Landegren et al., Science 241, 1077 (1988) and Barringer et
al. Gene 89:117 (1990)), transcription amplification (Kwoh et al.,
Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315),
self-sustained sequence replication (Guatelli et al., Proc. Nat.
Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective
amplification of target polynucleotide sequences (U.S. Pat. No.
6,410,276), consensus sequence primed polymerase chain reaction
(CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase
chain reaction (AP-PCR) (U.S. Pat. Nos. 5, 413,909, 5,861,245) and
nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.
Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is
incorporated herein by reference). Other amplification methods that
may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810,
4,988,617 and in U.S. Ser. No. 09/854,317, each of which is
incorporated herein by reference.
[0023] Additional methods of sample preparation and techniques for
reducing the complexity of a nucleic sample are described in Dong
et al., Genome Research 11, 1418 (2001), in U.S. Pat. No.
6,361,947, 6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491
(U.S. Patent Application Publication 20030096235), U.S. Ser. No.
09/910,292 (U.S. Patent Application Publication 20030082543), and
U.S. Ser. No. 10/013,598.
[0024] Methods for conducting polynucleotide hybridization assays
have been well developed in the art. Hybridization assay procedures
and conditions will vary depending on the application and are
selected in accordance with the general binding methods known
including those referred to in: Maniatis et al. Molecular Cloning:
A Laboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y, 1989);
Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to
Molecular Cloning Techniques (Academic Press, Inc., San Diego,
Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods
and apparatus for carrying out repeated and controlled
hybridization reactions have been described in U.S. Pat. Nos.
5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of
which are incorporated herein by reference
[0025] The present invention also contemplates signal detection of
hybridization between ligands in certain preferred embodiments. See
U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;
5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;
6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCT
Application PCT/US99/06097 (published as WO99/47964), each of which
also is hereby incorporated by reference in its entirety for all
purposes.
[0026] Methods and apparatus for signal detection and processing of
intensity data are disclosed in, for example, U.S. Pat. Nos.
5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758;
5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555,
6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S.
Ser. Nos. 10/389,194, 60/493,495 and in PCT Application
PCT/US99/06097 (published as WO99/47964), each of which also is
hereby incorporated by reference in its entirety for all
purposes.
[0027] The practice of the present invention may also employ
conventional biology methods, software and systems. Computer
software products of the invention typically include computer
readable medium having computer-executable instructions for
performing the logic steps of the method of the invention. Suitable
computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM,
hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The
computer executable instructions may be written in a suitable
computer language or combination of several languages. Basic
computational biology methods are described in, for example Setubal
and Meidanis et al., Introduction to Computational Biology Methods
(PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif,
(Ed.), Computational Methods in Molecular Biology, (Elsevier,
Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:
Application in Biological Science and Medicine (CRC Press, London,
2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide
for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd
ed., 2001). See U.S. Pat. No. 6,420,108.
[0028] The present invention may also make use of various computer
program products and software for a variety of purposes, such as
probe design, management of data, analysis, and instrument
operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729,
5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127,
6,229,911 and 6,308,170.
[0029] Additionally, the present invention may have preferred
embodiments that include methods for providing genetic information
over networks such as the Internet as shown in U.S. Ser. Nos.
10/197,621, 10/063,559 (United States Publication No. 20020183936),
U.S. Ser. No. 10/065,856, 10/065,868, 10/328,818, 10/328,872,
10/423,403, and 60/482,389.
[0030] b) Definitions
[0031] An "array" represents an intentionally created collection of
molecules which can be prepared either synthetically or
biosynthetically. In particular, the term "array" herein means an
intentionally created collection of probes (as used herein
typically polymers, peptides, polynucleotides and or
oligonucleotides) attached to at least a first surface of at least
one solid support wherein the identity of each polynucleotide at a
given predefined region or positionally defined location is known.
The terms "array," "biological chip" and "chip" are used
interchangeably. A polymer array also can include only a subset of
the complete set of probes. Similarly, a given array can exist on
more than one separate substrate, e.g., where the number of
sequences necessitates a larger surface area or more than one solid
substrate in order to include all of the desired oligonucleotide
sequences.
[0032] "Solid support," "support," and "substrate" refer to a
material or group of materials having a rigid or semi-rigid surface
or surfaces. In many embodiments, at least one surface of the solid
support will be substantially flat, although in some embodiments it
may be desirable to physically separate synthesis regions for
different compounds with, for example, wells, raised regions, pins,
etched trenches, or the like. According to other embodiments, the
solid support(s) will take the form of beads, resins, gels,
microspheres, fibers or other geometric configurations.
[0033] The phrase "coupled to a support" means bound directly or
indirectly thereto including attachment by covalent binding,
hydrogen bonding, ionic interaction, hydrophobic interaction, or
otherwise.
[0034] "Nucleotide" and "ribonucleotide" refers to both naturally
occurring and non-naturally occurring compounds having a
heterocyclic base, a sugar, and a linking group, preferably a
phosphate ester. For example, structural groups may be added to the
ribosyl or deoxyribosyl unit of the nucleotide, such as a methyl or
allyl group at the 2'-O position or a fluoro group that substitutes
for the 2'-O group. The linking group, such as a phosphodiester, of
the nucleic acid may be substituted or modified, for example with
methyl phosphonates or O-methyl phosphates. Bases and sugars can
also be modified, as is known in the art. For example, unless
otherwise limited the phrase would also cover synthetic and
naturally occurring variants of nucleic acids, including without
limitation, base variants such as 7-deazapurine,
8-aza-7-deazapurine, isocytosine, pseudo isocytosine, and
isouracil.
[0035] The terms "nucleic acid" or "nucleic acid molecule" as used
herein refer to a deoxyribonucleotide or ribonucleotide (see above)
polymer in either single or double stranded form. These terms also
encompass DNA-RNA hybrids.
[0036] The term "sugar" as used herein relates to monosaccharide
moieties. Preferred sugars are in cyclic form, for example, in
furanose (5-membered ring) or pyranose (6-membered ring) forms.
Sugars may be in any of their enantiomeric, diasteriomeric or
stereoisomeric forms.
[0037] As used herein, the terms "oligonucleotide" and
"polynucleotide" are used interchangeably in the conventional sense
to refer to molecules comprising two or more nucleosides, each
nucleoside being linked to at least one other nucleoside by an
internucleoside linkage. The oligonucleotides of the present
invention may be linear, branched, or cyclic, but are preferably
linear.
[0038] The term "mature oligonucleotide" or "full length
oligonucleotide" refers to an oligonucleotide which has
successfully undergone each of its coupling reactions so that its
sequence is complete and as expected per the synthetic scheme.
[0039] The term "prematurely terminated" or "truncated" refers to
an oligonucleotide which has failed at one or more coupling
reactions so that its sequence lacks specific units intended per
the synthetic scheme.
[0040] The term "reactive functional groups" refers to functional
groups which may react with other available functional groups under
specified conditions to yield a covalent linkage. Examples of
preferred reactive functional groups are hydroxyl (i.e., --OH) and
phosphoramidite (i.e., --OP(OR')NR.sub.2 wherein R' and R are
organic groups comprising 1 to 20 carbon atoms). A preferred group
of phosphoramidite functional groups are those for which R' is
--CH.sub.3, --CH.sub.2CH.sub.3, --CH.sub.2CH.sub.2CN, or
--C.sub.6H.sub.4Cl; and R is --CH(CH.sub.3).sub.2.
[0041] The term "protected" or "otherwise unreactive" functional
groups refers to functional groups which are essentially unreactive
toward other available functional groups under specified
conditions. The term "functional group protection" is used herein
in the conventional chemical sense to refer to common chemical
methods employed to reversibly render unreactive a functional
group, which otherwise would be reactive, under specified
conditions (such as pH, temperature, radiation, solvent, and the
like). A wide variety of such "protecting", "blocking", or
"masking" methods are widely used and well known in organic
synthesis. For example, a compound which has two non-equivalent
reactive functional groups, both of which would be reactive under
specified conditions, may be derivatized to render one of the
functional groups "protected", and therefore unreactive, under the
specified conditions; so protected, the compound may be used as a
reactant which has effectively only one reactive functional group.
After the desired reaction (involving the reactive functional
group) is complete, the protected group may be "deprotected" to
return it to its original functionality.
[0042] A wide variety of protecting group strategies are known. For
example, hydroxyl groups (i e., --OH) which are reactive toward a
certain other functional groups (for example, phosphoramidite)
under alkaline conditions might be "protected" by conversion to a
suitable ether, which is unreactive under alkaline conditions. When
it is desired to "deprotect" the hydroxyl group, the protected
compound might be treated with acid. For example, an --OH group may
be protected by reaction with DMT-Cl to yield the acid-labile
--ODMT group which may be deprotected, for example, by treatment
with a suitable acid, such as dichloroacetic acid.
[0043] Methods of obtaining nucleic acid sequences of a given
length and known sequence are known to those of skill in the art.
Methods of solid phase oligonucleotide synthesis are described in,
for example: Advances in the Synthesis of Oligonucleotides by the
Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P.,
Tetrahedron, 1992, 48, 2223-2311; U.S. Pat. Nos. 4,58,066;
4,500,707; 5,132,418; 4,973,679; 4,415,732; Re. 34,069; and
5,026,838.
[0044] The term "linker" means a molecule or group of molecules
attached to a substrate and spacing a synthesized polymer from the
substrate for exposure/binding to a receptor.
[0045] The term "activation energy wavelength" refers to that
wavelength of electromagnetic radiation that will activate a
photoprotective group or photocleavable group.
[0046] The term "activator" refers to a compound that facilitates
coupling of one nucleic acid to another, preferably in 3'-position
of one nucleic acid to 5'-position of the other nucleic acid or
vice a versa.
[0047] The terms "quality," "performance" and "intensity" are used
interchangeably herein when referring to oligonucleotide probes or
binding of a target molecule to oligonucleotide probes mean
sensitivity of oligonucleotide probes to bind to a target molecule
while giving a minimum of false signals.
[0048] The term "wafer" generally refers to a substantially flat
sample of substrate (i.e., solid-support) from which a plurality of
individual arrays or chips can be fabricated.
[0049] The term "functional group" means a reactive chemical moiety
present on a given monomer, polymer, linker or substrate surface.
Examples of functional groups include, e.g., the 3' and 5' hydroxyl
groups of nucleotides and nucleosides, as well as the reactive
groups on the nucleobases of the nucleic acid monomers, e.g., the
exocyclic amine group of guanosine, as well as amino and carboxyl
groups on amino acid monomers.
[0050] The term photoprotecting group (also called photolabile
protecting groups or photogroup for short) means a material which
is chemically bound to a reactive functional group on a monomer
unit, linker, or polymer and which may be removed upon selective
exposure to electromagnetic radiation or light, especially
ultraviolet and visible light.
[0051] The term "reactive group" refers to a group that allows a
covalent reaction to occur between for example a monomer and a
linker or between a second monomer and a first attached monomer. A
reactive group may be protected by photoprotective removable group.
Removal of the photogroup, yields a deprotected reactive group. The
terms "array" and "chip" are used interchangeably herein and refer
to the final product of the individual array of nucleic acid or
oligonucleotide sequences, having a plurality of positionally
distinct oligonucleotide sequences coupled to the surface of the
substrate. "Array" is used with reference to nucleic acid or
oligonucleotide, but it should be appreciated that either can
be-attached to a solid support. Reference will be made to
olinonucleotide arrays as a preferred example of the present
invention.
[0052] The term "alkyl" refers to a branched or straight chain
acyclic, monovalent saturated hydrocarbon radical of one to twenty
carbon atoms. The term "alkenyl" refers to an unsaturated
hydrocarbon radical which contains at least one carbon-carbon
double bond and includes straight chain, branched chain and cyclic
radicals.
[0053] The term "alkynyl" refers to an unsaturated hydrocarbon
radical which contains at least one carbon-carbon triple bond and
includes straight chain, branched chain and cyclic radicals.
[0054] The term "aryl" refers to an aromatic monovalent carboxylic
radical having a single ring (e.g., phenyl) or two condensed rings
(e.g., naphthyl), which can optionally be mono-, di-, or
tri-substituted, independently, with alkyl, lower-alkyl,
cycloalkyl, ydroxylower-alkyl, aminoloweralkyl, hydroxyl, thiol,
amino, halo, nitro, lower-alkylthio, lower-alkoxy,
mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl,
lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl,
lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano,
tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and
di-lower-alkylcarbamoyl.
[0055] Alternatively, two adjacent positions of the aromatic ring
may be substituted with a methylenedioxy or ethylenedioxy group.
The term "heteroaromatic" refers to an aromatic monovalent mono- or
poly-cyclic radical having at least one heteroatom within the ring,
e.g., nitrogen, oxygen or sulfur, wherein the aromatic ring can
optionally be mono-, di- or tri-substituted, independently, with
alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,
aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro,
lower-alkylthio, loweralkoxy, mono-lower-alkylamino,
di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl,
hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl,
lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl,
loweralkylcarbamoyl, and di-lower-alkylcarbamoyl. For example,
typical heteroaryl groups with one or more nitrogen atoms are
tetrazoyl, pyridyl (e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl),
pyrrolyl (e.g., 2-pyrrolyl, 2-(N-alkyl)pyrrolyl), pyridazinyl,
quinolyl (e.g. 2-quinolyl, 3-quinolyl etc.), imidazolyl,
isoquinolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridonyl or
pyridazinonyl; typical oxygen heteroaryl radicals with an oxygen
atom are 2-furyl, 3-furyl or benzofuranyl; typical sulfur
heteroaryl radicals are thienyl, and benzothienyl; typical mixed
heteroatom heteroaryl radicals are furazanyl and
phenothiazinyl.
[0056] Further the term also includes instances where a heteroatom
within the ring has been oxidized, such as, for example, to form an
N-oxide or sulfone. The term "optionally substituted" refers to the
presence or lack thereof of a substituent on the group being
defined. When substitution is present the group may be mono-, di-
or tri-substituted, independently, with alkyl, lower-alkyl,
cycloalkyl, hydroxylower-alkyl, aminoloweralkyl, hydroxyl, thiol,
amino, halo, nitro, lower-alkylthio, lower-alkoxy,
mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl,
lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl,
lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano,
tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and
di-lower-alkylcarbamoyl. Typically, electron-donating substituents
such as alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,
aminolower- alkyl, hydroxyl, thiol, amino, halo, lower-alkylthio,
lower-alkoxy, mono-lower-alkylamino and di-lower-alkylamino are
preferred.
[0057] The term "electron donating group" refers to a radical group
that has a lesser affinity for electrons than a hydrogen atom would
if it occupied the same position in the molecule. For example,
typical electron donating groups are hydroxy, alkoxy (e.g.
methoxy), amino, alkylamino and dialkylamine.
[0058] The term "leaving group" means a group capable of being
displaced by a nucleophile in a chemical reaction, for example
halo, nitrophenoxy, pentafluorophenoxy, alkyl sulfonates (e.g.,
methanesulfonate), aryl sulfonates, phosphates, sulfonic acid,
sulfonic acid salts, and the like.
[0059] "Activating group" refers to those groups which, when
attached to a particular functional group or reactive site, render
that site more reactive toward covalent bond formation with a
second functional group or reactive site. The group of activating
groups which are useful for a carboxylic acid include simple ester
groups and anhydrides. The ester groups include alkyl, aryl and
alkenyl esters and in particular such groups as 4-nitrophenyl,
N-hydroxylsuccinimide and pentafluorophenol. Other activating
groups are known to those of skill in the art.
[0060] A "cleavable moiety" or "releasable group" refers to a
molecule which can be cleaved or released under a set of distinct
conditions, e.g., certain wave lengths of light or certain chemical
conditions. As employed in the context of the present invention of
arrays of releasable polymer the conditions much be such as not to
substantially damage or harm the polymer in questions. Persons of
skill in the art will recognize what cleavable moiety may be
employed for example where the polymer is a nucleic acid or a
peptide.
[0061] "Predefined region" refers to a localized area on a solid
support. It can be where synthesis takes place or where a nucleic
acid is placed. Predefined region can also be defined as a
"selected region." The predefined region may have any convenient
shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
For the sake of brevity herein, "predefined regions" are sometimes
referred to simply as "regions." In some embodiments, a predefined
region and, therefore, the area upon which each distinct compound
is synthesized or placed is smaller than about 1 cm2 or less than 1
mm2. Within these regions, the molecule therein is preferably in a
substantially pure form. In additional embodiments, a predefined
region can be achieved by physically separating the regions (i.e.,
beads, resins, gels, etc.) into wells, trays, etc.
[0062] A "linker" is a molecule or group of molecules attached to a
substrate and spacing a synthesized polymer from the substrate for
exposurebinding to a receptor.
[0063] A "channel block" is a material having a plurality of
grooves or recessed regions on a surface thereof. The grooves or
recessed regions may take on a variety of geometric configurations,
including but not limited to stripes, circles, serpentine paths, or
the like. Channel blocks may be prepared in a variety of manners,
including etching silicon blocks, molding or pressing polymers,
etc.
[0064] A "monomer" is a member of the set of small molecules which
can be joined together to form a polymer. The set of monomers
includes but is not restricted to, for example, the set of common
L-amino acids, the set of common D-amino acids, the set of
synthetic amino acids, the set of nucleotides and the set of
pentoses and hexoses. As used herein, monomer refers to any member
of a basis set for synthesis of a polymer. Thus, monomers refers to
dimmers, trimers, tetramers and higher units of molecules which can
be joined to form a polymer. For example, dimmers of the 20
naturally occurring L-amino acids for a basis set of 400 monomers
for synthesis of polypeptides. Different basis sets of monomers may
be used at successive steps in the synthesis of a polymer.
Furthermore, each of the sets may include protected members which
are modified after synthesis.
[0065] A "polymer" is composed of two or more joined monomers and
includes for example both linear and cyclic polymers of nucleic
acids, polysaccharides, phospholipids, and peptides having either
a-, 0-, and o-amino acids, hetero-polymers in which a known drug is
covalently bound to any of the above, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or
other polymers.
[0066] A "releasable group" is a moiety or chemical group which is
labile, i.e., may be activated or cleaved, under a given set of
conditions, but is stable under other sets of conditions.
[0067] The term "monomer" as used herein refers to a single unit of
polymer, which can be linked with the same or other monomers to
form a biopolymer (for example, a single amino acid or nucleotide
with two linking groups one or both of which may have removable
protecting groups) or a single unit which is not part of a
biopolymer. Thus, for example, a nucleotide is a monomer within an
oligonucleotide polymer, and an amino acid is a monomer within a
protein or peptide polymer; antibodies, antibody fragments,
chromosomes, plasmids, mRNA, cRNA, tRNA etc., for example, are also
polymers.
[0068] The term "biopolymer" or sometimes refer by "biological
polymer" as used herein is intended to mean repeating units of
biological or chemical moieties. Representative biopolymers
include, but are not limited to, nucleic acids, oligonucleotides,
amino acids, proteins, peptides, hormones, oligosaccharides,
lipids, glycolipids, lipopolysaccharides, phospholipids, synthetic
analogues of the foregoing, including, but not limited to, inverted
nucleotides, peptide nucleic acids, Meta-DNA, and combinations of
the above. It is important to note that biopolymers and polymers
are not mutually exclusive. Proteins, enzymes, DNA, polyethylene,
RNA, are all polymers as they are derived from a repeating monomer
units. However, proteins, enzymes, DNA are all biopolymers as many
of them first appeared in nature. Sometimes, it is not easy to
classify something as a biopolymer or a polymer. For example, vast
number of human made amino acid derivatives and nucleotide
derivatives have been created and polymerized. Some of these are
based on natural products, many more are not. At this point the
distinction between the two can be somewhat semantical.
[0069] The term "biopolymer synthesis" as used herein is intended
to encompass the synthetic production, both in situ (in the cell)
and synthetically, e.g. by organic synthetic techniques outside of
the cell, of a biopolymer. Related to a bioploymer is a
"biomonomer".
[0070] The term "combinatorial synthesis strategy" as used herein
refers to a combinatorial synthesis strategy is an ordered strategy
for parallel synthesis of diverse polymer sequences by sequential
addition of reagents which may be represented by a reactant matrix
and a switch matrix, the product of which is a product matrix. A
reactant matrix is a 1 column by m row matrix of the building
blocks to be added. The switch matrix is all or a subset of the
binary numbers, preferably ordered, between 1 and m arranged in
columns. A "binary strategy" is one in which at least two
successive steps illuminate a portion, often half, of a region of
interest on the substrate. In a binary synthesis strategy, all
possible compounds which can be formed from an ordered set of
reactants are formed. In most preferred embodiments, binary
synthesis refers to a synthesis strategy which also factors a
previous addition step. For example, a strategy in which a switch
matrix for a masking strategy halves regions that were previously
illuminated, illuminating about half of the previously illuminated
region and protecting the remaining half (while also protecting
about half of previously protected regions and illuminating about
half of previously protected regions). It will be recognized that
binary rounds may be interspersed with non-binary rounds and that
only a portion of a substrate may be subjected to a binary scheme.
A combinatorial "masking" strategy is a synthesis which uses light
or other spatially selective deprotecting or activating agents to
remove protecting groups from materials for addition of other
materials such as amino acids.
[0071] The term "complementary" as used herein refers to the
hybridization or base pairing between nucleotides or nucleic acids,
such as, for instance, between the two strands of a double stranded
DNA molecule or between an oligonucleotide primer and a primer
binding site on a single stranded nucleic acid to be sequenced or
amplified. Complementary nucleotides are, generally, A and T (or A
and U), or C and G. Two single stranded RNA or DNA molecules are
said to be complementary when the nucleotides of one strand,
optimally aligned and compared and with appropriate nucleotide
insertions or deletions, pair with at least about 80% of the
nucleotides of the other strand, usually at least about 90% to 95%,
and more preferably from about 98 to 100%. Alternatively,
complementarity exists when an RNA or DNA strand will hybridize
under selective hybridization conditions to its complement.
Typically, selective hybridization will occur when there is at
least about 65% complementary over a stretch of at least 14 to 25
nucleotides, preferably at least about 75%, more preferably at
least about 90% complementary. See, M. Kanehisa Nucleic Acids Res.
12:203 (1984), incorporated herein by reference.
[0072] The term "copolymer" refers to a polymer that is composed of
more than one monomer. Copolymers may be prepared by polymerizing
one or more monomers to provide a copolymer.
[0073] The term "detectable moiety" means a chemical group that
provides a signal. The signal is detectable by any suitable means,
including spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means. In certain
cases, the signal is detectable by 2 or more means.
[0074] The detectable moiety provides the signal either directly or
indirectly. A direct signal is produced where the labeling group
spontaneously emits a signal, or generates a signal upon the
introduction of a suitable stimulus. Radiolabels, such as .sup.3H,
.sup.125I, .sup.35S, .sup.14C or .sup.32P, and magnetic particles,
such as Dynabeads.TM., are nonlimiting examples of groups that
directly and spontaneously provide a signal. Labeling groups that
directly provide a signal in the presence of a stimulus include the
following nonlimiting examples: colloidal gold (40-80 nm diameter),
which scatters green light with high efficiency; fluorescent
labels, such as fluorescein, Texas red, Rhoda mine, and green
fluorescent protein (Molecular Probes, Eugene, Oreg.), which absorb
and subsequently emit light; chemiluminescent or bioluminescent
labels, such as luminol, lophine, acridine salts and luciferins,
which are electronically excited as the result of a chemical or
biological reaction and subsequently emit light; spin labels, such
as vanadium, copper, iron, manganese and nitroxide free radicals,
which are detected by electron spin resonance (ESR) spectroscopy;
dyes, such as quinoline dyes, triarylmethane dyes and acridine
dyes, which absorb specific wavelengths of light; and colored glass
or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
See U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149 and 4,366,241.
[0075] A detectable moiety provides an indirect signal where it
interacts with a second compound that spontaneously emits a signal,
or generates a signal upon the introduction of a suitable stimulus.
Biotin, for example, produces a signal by forming a conjugate with
streptavidin, which is then detected. See Hybridization With
Nucleic Acid Probes. In Laboratory Techniques in Biochemistry and
Molecular Biology; Tijssen, P., Ed.; Elsevier: New York, 1993; Vol.
24. An enzyme, such as horseradish peroxidase or alkaline
phosphatase, that is attached to an antibody in a
label-antibody-antibody as in an ELISA assay, also produces an
indirect signal.
[0076] A preferred detectable moiety is a fluorescent group.
Fluorescent groups typically produce a high signal to noise ratio,
thereby providing increased resolution and sensitivity in a
detection procedure. Preferably, the fluorescent group absorbs
light with a wavelength above about 300 nm, more preferably above
about 350 nm, and most preferably above about 400 nm. The
wavelength of the light emitted by the fluorescent group is
preferably above about 310 nm, more preferably above about 360 nm,
and most preferably above about 410 nm.
[0077] The fluorescent detectable moiety is selected from a variety
of structural classes, including the following nonlimiting
examples: 1- and 2-aminonaphthalene, p,p'diaminostilbenes, pyrenes,
quaternary phenanthridine salts, 9-aminoacridines,
p,p'-diaminobenzophenone imines, anthracenes, oxacarbocyanine,
marocyanine, 3-aminoequilenin, perylene, bisbenzoxazole,
bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,
bis-3-aminopridinium salts, hellebrigenin, tetracycline,
sterophenol, benzimidazolyl phenylamine, 2-oxo-3-chromen, indole,
xanthen, 7-hydroxycoumarin, phenoxazine, salicylate,
strophanthidin, porphyrins, triarylmethanes, flavin, xanthene dyes
(e.g., fluorescein and rhodamine dyes); cyanine dyes;
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes and fluorescent
proteins (e.g., green fluorescent protein, phycobiliprotein).
[0078] A number of fluorescent compounds are suitable for
incorporation into the present invention. Nonlimiting examples of
such compounds include the following: dansyl chloride;
fluoresceins, such as 3,6-dihydroxy-9-phenylxanthhydrol;
rhodamineisothiocyanate; N-phenyl-1-amino-8-sulfonatonaphthalene;
N-phenyl-2-amino-6-sulfonatonaphthanlene;
4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid;
pyrene-3-sulfonic acid; 2-toluidinonapththalene-6-sulfonate;
N-phenyl, N-methyl 2-aminonaphthalene-6-sulfonate; ethidium
bromide; stebrine; auromine-0,2-(9'-anthroyl)palmitate; dansyl
phosphatidylethanolamin; N,N'-dioctadecyl oxacarbocycanine;
N,N'-dihexyl oxacarbocyanine; merocyanine, 4-(3'-pyrenyl)butryate;
d-3-aminodesoxy-equilenin; 12-(9'-anthroyl)stearate;
2-methylanthracene; 9-vinylanthracene;
2,2'-(vinylene-p-phenylene)bisbenzoxazole;
p-bis[2-(4-methyl-5-phenyl oxazolyl)]benzene;
6-dimethylamino-1,2-benzophenzin; retinol;
bis(3'-aminopyridinium)-1,10-decandiyl diiodide;
sulfonaphthylhydrazone of hellibrienin; chlorotetracycline;
N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;
N-[p-(2-benzimidazolyl)phenyl]maleimide;
N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin;
4-chloro-7-nitro-2,1,3-benzooxadizole; merocyanine 540; resorufin;
rose bengal and 2,4-diphenyl-3(2H)-furanone. Preferably, the
fluorescent detectable moiety is a fluorescein or rhodamine
dye.
[0079] Another preferred detectable moiety is colloidal gold. The
colloidal gold particle is typically 40 to 80 nm in diameter. The
colloidal gold may be attached to a labeling compound in a variety
of ways. In one embodiment, the linker moiety of the nucleic acid
labeling compound terminates in a thiol group (--SH), and the thiol
group is directly bound to colloidal gold through a dative bond.
See Mirkin et al. Nature 1996, 382, 607-609. In another embodiment,
it is attached indirectly, for instance through the interaction
between colloidal gold conjugates of antibiotin and a biotinylated
labeling compound. The detection of the gold labeled compound may
be enhanced through the use of a silver enhancement method. See
Danscher et al. J. Histotech 1993, 16, 201-207.
[0080] The term "effective amount" as used herein refers to an
amount sufficient to induce a desired result.
[0081] Although generally used herein to define separate regions
containing differing polymer sequences, the term "feature"
generally refers to any element, e.g., region, structure or the
like, on the surface of a substrate. Preferably, substrates will
have small feature sizes, and consequently, high feature densities
on substrate surfaces. For example, individual features will
typically have at least one of a length or width dimension that is
no greater than 100 microns, and preferably, no greater than 50
microns, and more preferably no greater than about 20 microns.
Preferred embodiments of the present invention may have features as
small as 1 micron, down to 0.5 microns. Thus, for embodiments
employing substrates having a plurality of polymer sequences on
their surfaces, each different polymer sequence will typically be
substantially contained within a single feature.
[0082] The term "fragmentation" refers to the breaking of nucleic
acid molecules into smaller nucleic acid fragments. In certain
embodiments, the size of the fragments generated during
fragmentation can be controlled such that the size of fragments is
distributed about a certain predetermined nucleic acid length.
[0083] The term "genome" as used herein is all the genetic material
in the chromosomes of an organism. DNA derived from the genetic
material in the chromosomes of a particular organism is genomic
DNA. A genomic library is a collection of clones made from a set of
randomly generated overlapping DNA fragments representing the
entire genome of an organism.
[0084] The term "hybridization" as used herein refers to the
process in which two single-stranded polynucleotides bind
non-covalently to form a stable double-stranded polynucleotide;
triple-stranded hybridization is also theoretically possible. The
resulting (usually) double-stranded polynucleotide is a "hybrid."
The proportion of the population of polynucleotides that forms
stable hybrids is referred to herein as the "degree of
hybridization." Hybridizations are usually performed under
stringent conditions, for example, at a salt concentration of no
more than 1 M and a temperature of at least 25.degree. C. For
example, conditions of 5.times. SSPE (750 mM NaCl, 50 mM
NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30.degree.
C. are suitable for allele-specific probe hybridizations. For
stringent conditions, see, for example, Sambrook, Fritsche and
Maniatis. "Molecular Cloning A laboratory Manual" 2.sup.nd Ed. Cold
Spring Harbor Press (1989) which is hereby incorporated by
reference in its entirety for all purposes above.
[0085] The term "hybridization conditions" as used herein will
typically include salt concentrations of less than about 1M, more
usually less than about 500 mM and preferably less than about 200
mM. Hybridization temperatures can be as low as 5.degree. C., but
are typically greater than 22.degree. C., more typically greater
than about 30.degree. C., and preferably in excess of about
37.degree. C. Longer fragments may require higher hybridization
temperatures for specific hybridization. As other factors may
affect the stringency of hybridization, including base composition
and length of the complementary strands, presence of organic
solvents and extent of base mismatching, the combination of
parameters is more important than the absolute measure of any one
alone.
[0086] The term "hybridization probes" as used herein are
oligonucleotides capable of binding in a base-specific manner to a
complementary strand of nucleic acid. Such probes include peptide
nucleic acids, as described in Nielsen et al., Science 254,
1497-1500 (1991), and other nucleic acid analogs and nucleic acid
mimetics.
[0087] The term "hybridizing specifically to" as used herein refers
to the binding, duplexing, or hybridizing of a molecule only to a
particular nucleotide sequence or sequences under stringent
conditions when that sequence is present in a complex mixture (for
example, total cellular) DNA or RNA.
[0088] The term "isolated nucleic acid" as used herein means an
object species invention that is the predominant species present
(i.e., on a molar basis it is more abundant than any other
individual species in the composition). Preferably, an isolated
nucleic acid comprises at least about 50, 80 or 90% (on a molar
basis) of all macromolecular species present. Most preferably, the
object species is purified to essential homogeneity (contaminant
species cannot be detected in the composition by conventional
detection methods).
[0089] The term "ligand" as used herein refers to a molecule that
is recognized by a particular receptor. The agent bound by or
reacting with a receptor is called a "ligand," a term which is
definitionally meaningful only in terms of its counterpart
receptor. The term "ligand" does not imply any particular molecular
size or other structural or compositional feature other than that
the substance in question is capable of binding or otherwise
interacting with the receptor. Also, a ligand may serve either as
the natural ligand to which the receptor binds, or as a functional
analogue that may act as an agonist or antagonist. Examples of
ligands that can be investigated by this invention include, but are
not restricted to, agonists and antagonists for cell membrane
receptors, toxins and venoms, viral epitopes, hormones (for
example, opiates, steroids, etc.), hormone receptors, peptides,
enzymes, enzyme substrates, substrate analogs, transition state
analogs, cofactors, drugs, proteins, and antibodies.
[0090] The term "mRNA," or sometimes referred to as "mRNA
transcripts," as used herein, includes, but not limited to pre-mRNA
transcript(s), transcript processing intermediates, mature mRNA(s)
ready for translation and transcripts of the gene or genes, or
nucleic acids derived from the mRNA transcript(s). Transcript
processing may include splicing, editing and degradation. As used
herein, a nucleic acid derived from an mRNA transcript refers to a
nucleic acid for whose synthesis the mRNA transcript or a
subsequence thereof has ultimately served as a template. Thus, a
cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA, a DNA amplified from the cDNA, an RNA transcribed from the
amplified DNA, etc., are all derived from the mRNA transcript and
detection of such derived products is indicative of the presence
and/or abundance of the original transcript in a sample. Thus, mRNA
derived samples include, but are not limited to, mRNA transcripts
of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA
transcribed from the cDNA, DNA amplified from the genes, RNA
transcribed from amplified DNA, and the like.
[0091] The term "nucleic acid library" or sometimes refer by
"array" as used herein refers to an intentionally created
collection of nucleic acids which can be prepared either
synthetically or biosynthetically and screened for biological
activity in a variety of different formats (for example, libraries
of soluble molecules; and libraries of oligos tethered to resin
beads, silica chips, or other solid supports). Additionally, the
term "array" is meant to include those libraries of nucleic acids
which can be prepared by spotting nucleic acids of essentially any
length (for example, from 1 to about 1000 nucleotide monomers in
length) onto a substrate. The term "nucleic acid" as used herein
refers to a polymeric form of nucleotides of any length, either
ribonucleotides, deoxyribonucleotides or peptide nucleic acids
(PNAs), that comprise purine and pyrimidine bases, or other
natural, chemically or biochemically modified, non-natural, or
derivatized nucleotide bases. The backbone of the polynucleotide
can comprise sugars and phosphate groups, as may typically be found
in RNA or DNA, or modified or substituted sugar or phosphate
groups. A polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. The sequence of
nucleotides may be interrupted by non-nucleotide components. Thus
the terms nucleoside, nucleotide, deoxynucleoside and
deoxynucleotide generally include analogs such as those described
herein. These analogs are those molecules having some structural
features in common with a naturally occurring nucleoside or
nucleotide such that when incorporated into a nucleic acid or
oligonucleoside sequence, they allow hybridization with a naturally
occurring nucleic acid sequence in solution. Typically, these
analogs are derived from naturally occurring nucleosides and
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be tailor made to
stabilize or destabilize hybrid formation or enhance the
specificity of hybridization with a complementary nucleic acid
sequence as desired.
[0092] The term "nucleic acids" as used herein may include any
polymer or oligomer of pyrimidine and purine bases, preferably
cytosine, thymine, and uracil, and adenine and guanine,
respectively. See Albert L. Lehninger, PRINCIPLES OF BIOCHEMISTRY,
at 793-800 (Worth Pub. 1982). Indeed, the present invention
contemplates any deoxyribonucleotide, ribonucleotide or peptide
nucleic acid component, and any chemical variants thereof, such as
methylated, hydroxymethylated or glucosylated forms of these bases,
and the like. The polymers or oligqmers may be heterogeneous or
homogeneous in composition, and may be isolated from
naturally-occurring sources or may be artificially or synthetically
produced. In addition, the nucleic acids may be DNA or RNA, or a
mixture thereof, and may exist permanently or transitionally in
single-stranded or double-stranded form, including homoduplex,
heteroduplex, and hybrid states.
[0093] The term "polymorphism" as used herein refers to the
occurrence of two or more genetically determined alternative
sequences or alleles in a population. A polymorphic marker or site
is the locus at which divergence occurs. Preferred markers have at
least two alleles, each occurring at frequency of greater than 1%,
and more preferably greater than 10% or 20% of a selected
population. A polymorphism may comprise one or more base changes,
an insertion, a repeat, or a deletion. A polymorphic locus may be
as small as one base pair. Polymorphic markers include restriction
fragment length polymorphisms, variable number of tandem repeats
(VNTR's), hypervariable regions, minisatellites, dinucleotide
repeats, trinucleotide repeats, tetranucleotide repeats, simple
sequence repeats, and insertion elements such as Alu. The first
identified allelic form is arbitrarily designated as the reference
form and other allelic forms are designated as alternative or
variant alleles. The allelic form occurring most frequently in a
selected population is sometimes referred to as the wildtype form.
Diploid organisms may be homozygous or heterozygous for allelic
forms. A diallelic polymorphism has two forms. A triallelic
polymorphism has three forms. Single nucleotide polymorphisms
(SNPs) are included in polymorphisms.
[0094] The term "primer" as used herein refers to a single-stranded
oligonucleotide capable of acting as a point of initiation for
template-directed DNA synthesis under suitable conditions for
example, buffer and temperature, in the presence of four different
nucleoside triphosphates and an agent for polymerization, such as,
for example, DNA or RNA polymerase or reverse transcriptase. The
length of the primer, in any given case, depends on, for example,
the intended use of the primer, and generally ranges from 15 to 30
nucleotides. However, longer primers are also preferred including
from 80 to 160 nucleotides. Short primer molecules generally
require cooler temperatures to form sufficiently stable hybrid
complexes with the template. A primer need not reflect the exact
sequence of the template but must be sufficiently complementary to
hybridize with such template. The primer site is the area of the
template to which a primer hybridizes. The primer pair is a set of
primers including a 5' upstream primer that hybridizes with the 5'
end of the sequence to be amplified and a 3' downstream primer that
hybridizes with the complement of the 3' end of the sequence to be
amplified.
[0095] The term "probe" is often used in the context of array
technology as the polymer bound to the array. See U.S. Pat. No.
6,582,908 for an example of arrays having all possible combinations
of probes with 10, 12, and more bases. Examples of probes that can
be investigated by this invention include, but are not restricted
to, agonists and antagonists for cell membrane receptors, toxins
and venoms, viral epitopes, hormones (for example, opioid peptides,
steroids, etc.), hormone receptors, peptides, enzymes, enzyme
substrates, cofactors, drugs, lectins, sugars, oligonucleotides,
nucleic acids, oligosaccharides, proteins, and monoclonal
antibodies. As used herein, probes may be designed to be
releasable, i.e., capable of being severed from the array and,
thus, as interchangeable "primers."
[0096] c) Massively Parallel Oligonucleotide Probe Synthesis and
Purification
[0097] According to one aspect of the present invention, massively
parallel oligonucleotide probe synthesis and purification is
provided for applications that utilize large collections of defined
high-fidelity oligonucleotides (e.g., from about 10.sup.1 to about
10.sup.5 different sequences, generally between 25-160 bases in
length) with or without authentic 3'-hydroxy termini. Preferably,
the oligonucleotides are between about 80 to 160. It is also
preferred that the oligonucleotides contain authentic 3' hydroxyl
groups. However, other types of 3' hydroxyl groups are also
preferred, such as phosphorylated.
[0098] In accordance with one aspect of the present invention,
photolithographic synthetic strategies may provide a convenient
approach to efficiently produce an array of such probes. In
accordance with an aspect of the present invention, synthesis may
be initiated with a reverse-orientation RNA monomer that contains
an orthogonal 2'-OH group with an appropriate protective group. Two
exemplary monomers are shown in FIG. 2. Following conventional
3'.fwdarw.5' oligonucleotide synthesis, the 2'-OH protecting group
is removed to allow base-induced intramolecular
transesterification. The transesterification reaction causes
release of the synthesized oligonucleotide, leaving an authentic
3'-hydroxy functionality, while the 2',3'-cyclic phosphate remains
attached to the solid support (see FIG. 1).
[0099] Accordingly, one embodiment of the present invention
provides a method of fabricating a plurality of oligonucleotides
having free 3'-hydroxyl groups from a high density oligonucleotide
array, said method comprising the steps of [0100] a) providing a
solid substrate comprising a plurality of ribonucleotides attached
thereto at a density, one said ribonucleotide shown below ##STR1##
wherein PG.sub.1 is protecting group 1, PG.sub.2 is protecting
group 2, B is a naturally or non-naturally occurring base, and said
ribonucleotide is attached to said substrate through the
5'-hydroxyl group; [0101] b) selectively removing PG.sub.1 in
pre-selected areas of the substrate to provide a plurality of free
3'-hydroxyl groups on said ribonucloetide; [0102] c) reacting said
free 3'-hydroxyl groups with a 2'-deoxyribonucleotide having the
structure ##STR2## wherein PG.sub.3 is protecting group 3 and RG is
a reactive group to couple said 2'-deoxyribonucleotide to said
ribonucleotide to provide the structure ##STR3## [0103] d)
selectively removing PG.sub.3 from the 5'-hydroxyl of said
2'-deoxyribonucleotide in pre-selected areas to provide a plurality
of free 5'-hydroxyl groups; [0104] e) reacting said free
5'-hydroxyl groups with an additional 2'-deoxyribonucleotide having
the structure ##STR4## to yield a product of the structure ##STR5##
[0105] f) repeating steps d and e one or more times to provide said
oligonucleotides attached to said solid substrate; [0106] g)
removing PG.sub.2 from one or more of said ribonucleotides to
provide a free 2'-hydroxyl group on each of said one or more
ribonucleotides; and [0107] h) transesterifying each of said one or
more ribonucleotides to yield said solid substrate having a cyclic
ester attached thereto and free oligonucleotides, each
oligonucleotide having a 3'-hydroxyl group and having the structure
##STR6##
[0108] In certain aspects of the present invention the
2'-deoxyribonucleotide has a phosphoramidite reactive group as
shown by ##STR7## wherein R.sub.1 is selected from cyanoethyl,
methyl, t-butyl, trimethylsilyl or the like, and R.sub.2 and
R.sub.3 are independently selected from isopropyl, cyclohexyl or
the like.
[0109] In certain aspects of the present invention the
oligonucleotides are probes while in other aspects of the present
invention the oligonucleotides are primers.
[0110] In one aspect of the present invention, PG.sub.1, PG.sub.3
and PG.sub.4 are independently selected protecting groups. These
protecting groups may be the same as or different from one another.
It has been discovered in accordance with the present invention
that to achieve suitable primer purity and quantity, a
highly-efficient photogroup (>90% average stepwise coupling
efficiency) is preferred for PG.sub.1, PG.sub.3 and PG.sub.4, such
as NPPOC or MBPMOC: ##STR8## Both NNPOC and MBPMOC give greater
than 90% stepwise coupling. For example NNPOC gives 97-98% stepwise
coupling.
[0111] Alternatively, DMT-based chemistry could be employed in
conjunction with photoacid. DMT-based resist methodologies have
been found, in accordance with the present invention to provide up
to a 99% stepwise yield. In certain embodiments of the present
invention, when PG.sub.1, PG.sub.3 or PG.sub.4 are DMT, the DMT
group may be removed in selected areas by exposure to acid
generated by a photoacid generator in the presence of electro
magnetic radiation of an appropriate wavelength in the presence of
an acid scavenger. In further embodiments of the present invention
the acid scavenger is selected from the group consisting of organic
bases and polymeric bases. In preferred aspects of the present
invention, the acid scavenger is a polymeric base.
[0112] In another aspect of the present invention, it is further
contemplated that combinations of DMT-based and photochemical-based
probe assembly could be performed, for example to assemble common
regions of the probe sequence. Despite the specific primer array
synthesis methodology, high-density substrates (200-2000
pmoles/cm.sup.2) can be employed to significantly boost primer
yield. Such substrates are typically based upon three-dimensional
architectures, thin-films or polymeric coatings.
[0113] The rate of transesterification (i.e., oligonucleotide
release) can be significantly enhanced by raising the pH of the
aqueous solution (pH 9-12) and/or by the addition of particular
metal ions, which are known in the art to facilitate or catalyze
such reactions.
[0114] Exemplary 2'-OH RNA monomer protecting groups, PG.sub.2, are
Ac (removed during base deprotection), FPMP or CEE (removed with
mild acid, but not strong acid), TBDMS or TOM (removed with
fluoride ions) or even a photogroup that is active at wavelengths
longer than 365 nm.
[0115] In preferred embodiments of the present invention PG.sub.2
will be orthogonal to PG.sub.1, PG.sub.3, and PG.sub.4. As such,
the conditions used to deprotect PG.sub.2 are different than those
used to deprotect PG.sub.1, PG.sub.3 and PG.sub.4. For example, if
acid labile protecting groups are used for PG.sub.1, PG.sub.3 and
PG.sub.4, then PG.sub.2 may be protected with a photolabile
protecting group. The converse is also true, i.e., if photolabile
protecting groups are used for PG.sub.1, PG.sub.3 and PG.sub.4,
then PG.sub.2 may be protected with an acid labile protecting
group. In some embodiments, photo labile protecting groups can be
used for all of PG.sub.1, PG.sub.2, PG.sub.3 and PG.sub.4, provided
that the photo labile protecting group of PG.sub.2 is active at
wavelengths longer than those of the photo labile protecting group
of PG.sub.1, PG.sub.3 and PG.sub.4, e.g., longer than 365 nm.
[0116] Because the coupling reactions between the free 5'-hydroxyl
groups and the reactive group of the 2'-deoxyribonucleotide
typically are not 100% efficient in a finite time period, a small
percentage of truncated sequences, i.e., sequences which did not
undergo the expected coupling event, is produced at each coupling
step. To prevent these truncated sequences from undergoing further
coupling reactions to produce unexpected oligonucleotide sequences,
the unreacted free 5'-hydroxyl group can be capped prior to
performing additional coupling reactions to render it unreactive
for subsequent synthesis steps. This capping reaction may be
accomplished by acetylation using methods known in the art.
[0117] In another aspect of the present invention, steps are
employed to enrich or purify the full-length oligonucleotides
(e.g., probes or primers). For example, if a base-stable RNA
monomer is employed, the final 5'-DMT group is retained on the
probe and the array is deprotected in the usual manner. The
deprotection step removes the DNA protecting groups, reverses some
unwanted chemical modifications, and also cleaves depurinated
sites. The protecting groups and truncated fragments are washed
away from the solid support, leaving behind the immobilized
full-length probes that contain a 5'-DMT group as well as
immobilized truncated species that do not possess a 5'-DMT
group.
[0118] A subsequent processing step causes removal of the 2'-OH RNA
protecting group and subsequent transesterification/release of
probes (FIG. 3). The solution containing the mixture of free
oligonucleotides can then be adsorbed onto a disposable hydrophobic
oligonucleotide purification cartridge (e.g., Waters Sep-Pak, ABI
OPC, etc.) to isolate only those oligonucleotide species that
possess a 5'-DMT group.
[0119] Accordingly, the present invention provides a method of
purifying a set of oligonucleotides, comprising the steps of [0120]
a) providing a solid substrate comprising a plurality of
ribonucleotides attached thereto at a density, one said
ribonucleotide shown below ##STR9## wherein PG.sub.1 is protecting
group 1, PG.sub.2 is an alkalai resistant protecting group, B is a
naturally or non-naturally occurring base, and said ribonucleotide
is attached to said substrate through the 5'-hydroxyl group; [0121]
b) selectively removing PG.sub.1 in pre-selected areas of the
substrate to provide a plurality of free 3'-hydroxyl groups on said
ribonucloetide; [0122] c) reacting said free 3'-hydroxyl groups
with a 2'-deoxyribonucleotide having the structure ##STR10##
wherein PG.sub.3 is DMT, B is a naturally or non-naturally
occurring base in which the exocyclic amine groups are protected
with alkalai labile protecting groups, and RG is a reactive group
to couple said 2'-deoxyribonucleotide to said ribonucleotide to
provide the structure ##STR11## [0123] d) selectively removing
PG.sub.3 from the 5'-hydroxyl of said 2'-deoxyribonucleotide in
pre-selected areas to provide a plurality of free 5'-hydroxyl
groups; [0124] e) reacting said free 5'-hydroxyl groups with an
additional 2'-deoxyribonucleotide having the structure ##STR12##
wherein PG.sub.4 is DMT, to yield a product of the structure
##STR13## [0125] f) repeating steps d and e one or more times to
provide said oligonucleotides attached to said solid substrate;
[0126] g) deprotecting said set of oligonucleotides while said
oligonucleotides are still attached to said substrate by subjecting
said oligonucleotides to alkaline conditions, wherein said alkaline
conditions remove said alkalai labile protecting groups acting to
protect said exocyclic amines and in addition cleave depurinated
DNA, leaving a 3'-end of the cleaved depurinated strand attached to
the substrate and releasing a truncated fragment; [0127] h) washing
the solid support to remove said released truncated fragments and
protecting groups, leaving full length oligonucleotides having a
DMT group on the 5'-hydroxyl group and truncated oligonucleotides
without the 5'-DMT group; [0128] i) removing PG.sub.2 from one or
more of said ribonucleotides to provide a free 2'-hydroxyl group on
each of said one or more ribonucleotides; [0129] j)
transesterifying each of said one or more ribonucleotides to yield
said solid substrate having a cyclic ester attached thereto and a
mixture of full length oligonucleotides having 5'-DMT groups and
free 3'-hydroxyl groups and having the structure ##STR14## and
truncated fragments lacking the DMT group; [0130] k) applying the
mixture to hydrophobic oligonucleotide purification resin to
isolate only those oligonucleotides having the 5'-DMT group to
yield full length oligonucleotides; and [0131] l) removing the
5'-DMT group to provide full length oligonucleotides having both
5'- and 3'-hydroxyl groups.
[0132] As an alternative to the procedure described above, the
2'-OH RNA protecting group is simply Ac, which is removed along
with the other protecting groups during standard deprotection. If
the non-catalyzed transesterification rate is low, then full-length
oligonucleotides will not be substantially released until the
reaction conditions are appropriately adjusted to cause
transesterification.
[0133] It is contemplated that reporter groups (e.g., chromophores,
fluoraphores, detectable labels) or affinity tags (e.g., biotin)
can be incorporated into the probe sequences, in either
single-color or multi-color formats. Phosphorylation at either
terminus (or both termini) is also possible. Dual-labeled
oligonucleotide "probes" (e.g., TaqMan probes and molecular
beacons) are also contemplated in accordance with the instant
invention. Additionally, non-conventional building blocks (e.g.,
nucleoside analogues or mimics) could be incorporated into the
probe/primer, either in part or in whole. Primer quantity will be a
function of the stepwise coupling yield, primer length, the surface
loading, feature size and feature redundancy of a given array
design. The relative concentration of each probe/primer can be
adjusted by controlling the redundancy of the array design.
[0134] It should be noted that many of the embodiments described
herein are described with reference to the fabrication of
oligonucleotide probes and/or primers. However, these descriptive
embodiments are not intended to limit the types of or uses for
oligonucleotides which can be produced and purified or isolated
using the described methods. The oligonucleotides produced by the
described methods can have any desired sequence composition and be
utilized in any context in which oligonucleotides of defined
sequence are desired. Oligonucleotides can be prepared according to
the methods of the invention singly or in sets within the larger
population of distinct sequences. By way of non-limiting example,
forward and reverse PCR primers for a particular target sequence
may be synthesized as a set on the array; alternatively, sets of
probes or primers differing by a single base at specific positions
can be produced.
[0135] A preferred aspect of the present invention is that
synthesis is initiated with a reverse-orientation RNA monomer that
contains an orthogonal 2'-OH protecting group. Following
conventional 3'.fwdarw.5' probe synthesis, the 2'-OH protecting
group is removed to allow base-induced intramolecular
transesterification. The transesterification reaction causes
release of the synthesized probe with an authentic 3'-hydroxy
functionality, while the 2',3'-cyclic phosphate remains attached to
the solid support (see FIG. 1). The rate of transesterification
(i.e., probe release) can be significantly enhanced by raising the
pH of the aqueous solution (pH 9-12) and/or by the addition of
particular metal ions, which are known in the art to facilitate or
catalyze such reactions.
[0136] Two exemplary monomers are depicted in FIG. 2: Exemplary
2'-OH RNA monomer protecting groups are Ac (removed during base
deprotection), FPMP or CEE (removed with mild acid, but not strong
acid), TBDMS or TOM (removed with fluoride ions) or even a
photogroup that is active at wavelengths longer than 365 nm.
[0137] Another preferred aspect of the present invention is the
steps that are employed to enrich or purify the full-length probes
(see FIG. 3). For example, if a base-stable RNA monomer is
employed, the final 5'-DMT group is retained on the probe and the
array is deprotected in the usual manner. The deprotection step
removes the DNA protecting groups, reverses some unwanted chemical
modifications, and also cleaves depurinated sites. The protecting
groups and truncated fragments are washed away from the solid
support, leaving behind the immobilized full-length probes that
contain a 5'-DMT group as well as truncated species that do not
possess a 5'-DMT group. A subsequent processing step causes removal
of the 2'-OH RNA protecting group and subsequent
transesterification/release of probes. The solution containing the
mixture of probes can then be adsorbed onto a disposable
hydrophobic oligonucleotide purification cartridge (e.g., Waters
Sep-Pak, ABI OPC, etc.) to isolate only those probe species that
possess a 5'-DMT group.
[0138] As an alternative to the procedure described above, the
2'-OH RNA protecting group is simply Ac, which is removed along
with the other protecting groups during standard deprotection. If
the non-catalyzed transesterification rate is low, then full-length
probes will not be substantially released until the reaction
conditions are appropriately adjusted to cause
transesterification.
[0139] It is contemplated that reporter groups (e.g., cluomophores,
fluorophores, detectable labels) or affinity tags (e.g.,biotin) can
be incorporated into the probe sequences, in either single-color or
multi-color formats. Phosphorylation at either terminus (or both
ternlini) is also possible. Dual-labeled oligonucleotide "probes"
(e.g., TaqMan probes and molecular beacons) are also contemplated.
Additionally, non-conventional building blocks (e.g., nucleoside
analogues or mimics) could be incorporated into the probe/primer,
either in part or in whole. Primer quantity will be a function of
the stepwise coupling yield, primer length, the surface loading,
feature size and feature redundancy of a given array design. The
relative concentration of each primer can be adjusted by
controlling the redundancy of the array design.
[0140] In accordance with an aspect of the present invention, a
method for massively parallel oligonucleotide probe synthesis and
release of said oligonucleotides from an array of probes on a solid
substrate is provided, the method having the steps of providing a
solid substrate; [0141] attaching a plurality of linkers to the
substrate, each said linker comprising a cleavable moiety, wherein
said cleavable moiety is activatable only at a distinct set of
conditions and wherein activation of said cleavable moiety disrupts
the linker to allow of a polymer bound to said linker, to provide a
substrate with a plurality of attached linkers; [0142] attaching a
first monomer to at least one of said plurality of attached linkers
to provide an attached first monomer; [0143] attaching a second
monomer to a least one of said attached first monomers or said
plurality of attached linkers to provide an attached second
monomor; [0144] attaching a third monomer to a least one of said
attached first monomer, said second monomer or said plurality of
attached linkers to provide an attached third monomer; repeating
said steps of attaching monomers until the desired array of
polymers is complete; [0145] and subjecting the array to the
distinct set of conditions to release polymers from said array.
[0146] In a preferred embodiment of the present invention, the
desired array has between 10.sup.1 to 10.sup.5 oligonucleotides of
different sequences which are between about 80-160 bases in
length.
[0147] According to another aspect of the present invention, it is
preferred that said released oligonucleotides have authentic
3'-hydroxy termini upon exposure to said distinct set of conditions
or are further processed to have authentic 3' hydroxyl termini.
[0148] According to another aspect of the presesent invention, it
is preferred that the method further comprising the use of the
photoprotective groups, preferably NPPOC or NNPOC to provide
suitable primer purity and quantity.
[0149] According to another aspect of the present invention, it is
preferred that the method employ DMT-based photoresist groups to
provide suitable primer purity and quantity.
[0150] According to another aspect of the present invention, it is
preferred that ink-jet based in situ oligonucleotide synthesis is
used to provide the oligonucleotide probes.
[0151] According to another aspect of the present invention, it is
preferred that the method is initiated with a reverse-orientation
RNA monomer that contains an orthogonal 2'-OH protecting group.
[0152] According to another aspect of the present invention, it is
preferred that following conventional 3'.fwdarw.5' probe synthesis,
the 2'-OH protecting group is removed to allow base-induced
intramolecular transesterification. Base-induced intramolecular
transesterification is preferentially performed by raising the pH
of the aqueous solution to pH 9-12 or by the addition of particular
metal ions.
[0153] All patents, patent applications, and literature cited in
the specification are hereby incorporated by reference in their
entirety. In the case of any inconsistencies, the present
disclosure, including any definitions therein will prevail.
[0154] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
* * * * *